Novel methods for HIV sequencing and genotyping

ABSTRACT

Methods are provided for amplifying regions of the HIV pol gene amplifying double-stranded nucleic acid template derived from HIV tube RT-PCR with novel PCR primers to produce amplified target sequences. Methods are also provided for analyzing the nucleotide sequence of these amplified targets using novel sequencing primers and the data is analyzed. The determined nucleotide sequence can be compared to the sequence of known drug resistance mutations in the HIV pot gene to determine the viral genotype.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates to methods for sequencing the HumanImmunodeficiency Virus (HIV) nucleic acids. More specifically, thepresent invention relates to methods for obtaining information on thegenetic sequences of HIV nucleic acid from a patient. That informationcan be used to genotype a HIV quasi-species present in the patient.

The detection of mutations conferring drug resistance in the HIV polgene is significant in determining drug sensitivity of the virus. Duringthe course of treatment of a disease, the infectious microorganism orvirus, such as HIV, can become resistant due to a loss of sensitivity tothe particular drug in use, which generally-results in spread of thedisease and increased morbidity. At the genetic level, important changescan occur within the virus in response to drug therapy. Specific changesin a nucleic sequence or nucleic acid sequences of the virus thatcorrelate with drug resistance are defined as drug resistance mutations.The nucleic sequences may be target nucleic acid sequences that areaffected by a drug or therapeutic agent. Such target nucleic acids mayencode a viral protein, such as an enzyme.

Due to the emergence of drug resistance mutations, one should obtaininformation concerning the genetic sequence of target nucleic acids ofthe virus in the patient for proper diagnosis and for choosing anappropriate treatment. Once this information is obtained, failure ofdrug therapy can be monitored at the genetic level rather than waitingfor the re-emergence or worsening of clinical symptoms. This may beaccomplished by isolating the nucleic acid for the infectious organism(virus) from the patient, determining the sequence of the target nucleicacids of the organism, and identifying mutations known to confer drugresistance.

This approach can also be used to intelligently prescribe effective drugtreatment. The nucleic acid sequence of the organism's target nucleicacids can be obtained from the patient prior to treatment, and theorganism's resistance to a particular drug can be determined.

One way to obtain de novo sequence information for the target nucleicacids of an HIV quasi-species present in a patient is to obtain a sampleof the patient's plasma or tissue. The viral RNA or DNA from that sampleis then extracted. If the genetic information is RNA, it shouldtypically be reverse-transcribed into DNA. The DNA of the HIV targetnucleic acid is then amplified by PCR, and the PCR products aresequenced. This sequence data can then be compared to a referencesequence for HIV and with all known drug resistance mutations.

Many RNA containing viruses, including HIV, rapidly mutate even in theabsence of drug therapy. This is due to the lack of fidelity andproof-reading functions by the virus's RNA polymerase or reversetranscriptase for retroviruses. For HIV reverse transcriptase, forexample, the estimated spontaneous mutation rate is 3×10⁻⁵ nucleotidesper replication cycle (Mansky and Temin, J. Virol., 69:5087-94, 1995).

The frequent use of antiviral drugs in the treatment of HIV infectionhas led to the development of drug resistance in AIDS patients. In thecase of HIV, is the genetic sequence of the HIV pot gene (which encodesthe viral protease and reverse transcriptase) is often the targetnucleic acid (Wainberg and Friedland, J. Am. Med. Assn., 279:1977-93,1998). Drug resistant HIV mutants have been isolated from infectedindividuals. The present inventors believe that a 1.57 kilobase (kb)region of the pol gene is a particularly important region containingclinically relevant mutations.

The high degree of enzyme-induced genetic variability, in addition tothe selective pressures of drug therapy, makes genotypic assessment ofHIV very complex. Typically, HIV infected individuals harbor multipleviral genotypes or quasi-species, whether due to random enzyme-inducedmutations, drug resistance-related mutations, or a combination of suchmutations. As drug resistant mutant HIV strains become more prevalent,individuals with no history of drug treatment are becoming infected withdrug resistant viruses.

Presently, determining appropriate treatment of HIV infections does nottypically involve genetic analysis of the HIV pol gene (protease andreverse transcriptase) from patient plasma HIV RNA. Thus, physicianstypically can only diagnose drug resistance in a patient if the patientfails to respond to therapy. Moreover, without genetic analysis, if apatient is failing therapy, it is difficult, if not impossible, todetermine for which drugs the patient is still sensitive. By isolating,amplifying, and sequencing the patient's HIV pol gene from plasma, itwill be possible to determine the number of drug resistance mutationsand tailor further therapy accordingly.

Consequently, there exists a need for rapid, reliable methods forobtaining the de novo nucleic acid sequences from clinical samples frompatients who are, or may be, infected with HIV. In addition to providingpatient-specific genotype information for use in identifying anappropriate treatment and monitoring drug resistance, the public healthcommunity would benefit from rapid, standardized, and reliable sequenceinformation to establish the significance and relevance ofdrug-associated resistance mutations.

Current HIV genotyping procedures include hybridization based assaysusing labeled oligonucleotide probes and “home brew” (internallycreated) sequencing based assays. Because of a high rate of mutation inHIV, technologies using labeled oligonucleotides to represent “mutant”or “wild-type” forms at a particular codon of the gene sequence will beadversely affected. For example, mutations which are not associated withdrug resistance will frequently occur, and may affect the binding ofeither “wild-type” or “mutant” probes, giving an anomalous result.Therefore, de novo sequencing should be a more accurate way to representgenetic changes of these highly variable sequences. This is especiallyimportant for organisms such as HIV because of the inherent geneticvariability due to the lack of proofreading activity of HIV reversetranscriptase. Since the understanding of HIV mutations and theirassociation with drug resistance is continually being elucidated,obtaining the de novo sequence of the HIV pol gene from patientpopulations undergoing drug therapy is important in establishing theclinical relevance of drug resistance in HIV.

Although a variety of home brew sequencing based assays have been usedin individual research labs, the present inventors are not aware ofcomprehensive, commercially available systems for determining the denovo sequence of infectious organisms. The general poor quality and lackof proper controls, seen with most home brew assays, have hinderedgeneration of accurate data which are crucial for studying drugresistance.

Other HIV genotyping procedures involve polymerase chain reaction (PCR)using nested primers to amplify HIV nucleic acid sequences. The nestedprimer procedure typically requires a different set of primers for eachPCR cycle, with each successive set of primers being selected to annealwithin the fragment amplified by the prior PCR cycle. While thisprocedure is effective at amplifying a known sequence present at lowcopy number, the use of multiple sets of primers, each of which musthybridize successively to a target sequence in the gene of interest, canresult in a loss of the ability to amplify highly variable nucleic acidsequences. This would occur,.for example, where a mutation was locatedin any of the target sequences in a region where primers are designed tohybridize. This results in biased selection of HIV quasi-species whereinsignificant drug resistance mutations may remain entirely undetecteduntil drug failure occurs in the patient.

One goal of HIV genotyping is to monitoring drug resistance at thegenetic level by identifying as many different HIV quasi-species aspossible. Among such quasi-species, there are likely to be mutations dueto drug resistance as well as random mutations due to polymerase errorin the virus population. The number of HIV quasi-species detectable bythe genotyping assay should be maximized. Therefore, a need also existsfor genotyping procedures which detects many different HIV quasi-speciesfrom a rapidly mutating virus population.

An object of the invention is to provide methods for obtaining de novosequence information for different HIV quasi-species present in patient3s samples. This invention will allow effective diagnosis and treatmentfor patients and will also provide methods for monitoring drug therapyfailures at the genetic level. Another object of the instant inventionis to provide a standardized assay which will allow for rapid andaccurate identification of mutations associated with drug resistance.

According to certain preferred embodiments, the inventors have achievedimproved sensitivity and determined a greater number of HIVquasi-species than procedures that employ nested primers. Certainembodiments involve a stream-lined assay, including a single-tube twostep amplification procedure, coupled with automated sequence analysisand correlation with known drug resistance mutations. Such embodimentsprovide rapid, reliable assays that have acceptable sensitivity andspecificity.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be apparent fromthe description, or may be learned by practice of the invention.

According to certain embodiments, the invention comprises methods foramplifying a target nucleic sequence of HIV including combining adouble-stranded nucleic acid template derived from HIV with certainspecific PCR primers, a temperature-stable DNA polymerase, anddeoxyribonucleotides, and amplifying the template to produce amplifiedtarget sequences. Certain embodiments include analysis of the amplifiedsequences. In certain embodiments, target sequence analysis isaccomplished using a computer program which determines target genesequences and, then compares those sequences with HIV referencesequences and a table of known drug resistance mutations.

In other embodiments, the invention comprises methods for sequencing HIVnucleic acid including combining a double-stranded nucleic acid templatederived from HIV and one or more specific sequencing primers, amplifyingthe HIV derived double-stranded nucleic acid template to produceamplified sequencing products, separating those amplified sequencingproducts to obtain nucleic acid sequence data, and analyzing the nucleicacid sequencing data.

In yet other embodiments of the present invention, target nucleic acidsequencing involves use of particular dye-terminator chemistry. Suchchemistry is useful for automated sequence analysis and determination ofheterozygosity at a given nucleotide.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

DESCRIPTION OF PREFERRED EMBODIMENTS

The references discussed or cited in this application are allspecifically incorporated by reference into this application.

For use in the present invention, typically, samples from infected orpotentially infected patients are used as a source of HIV. Tissuesamples or body fluid samples can be used. As used herein, body fluidsinclude, but are not limited to, whole blood, plasma, serum, peripheralblood mononuclear cells (PBMC), other fractionated blood products,tears, saliva, semen, vaginal secretions, serous and pleural cavityfluids, washes from various mucous membranes such as the eye, nose orthroat, and the like. Plasma, which may be obtained by methods wellknown in the art, is exemplary of a preferred source of proviral nucleicacid and will be used in the following examples.

According to certain embodiments, plasma samples, which may be frozenprior to use, are centrifuged under conditions that results in pelletingof the virion from the sample. After discarding the supernatant, thevirus is lysed with lysis buffer. A particular buffer that may be usedincludes 4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sodiumlauroyl sarcosine, 100 mM dithiothreitol, 80 μg/ml glycogen in sterilewater. Viral RNA can be precipitated using absolute isopropanol and canbe pelleted by centrifugation. The resulting supernatant is discardedand the pellet is washed and again pelleted by centrifugation. The washsolution can include 70% ethanol. HIV RNA samples are resuspended in anRNA diluent. According to certain embodiments, such a diluent caninclude 10 ng/ml polyrA from Pharmacia in RNase-free water. All stepscan be performed using prechilled instruments and buffers, and thesamples can be maintained at 4° C. or stored at −70° C.

Double stranded DNA template is then created from the prepared HIV RNA.According to certain embodiments, random hexamers are used as primers atthe 3′ end in conjunction with a reverse transcriptase (RT) procedure.An advantage of using random hexamers is that sequence variability willnot affect cDNA generation. Random hexamers may also stabilize RNAsecondary structure, which is known to be quite significant in HIV polRNA. Appropriate conditions are used that result in cDNA generation frompurified HIV RNA. In certain preferred embodiments, the double strandedtemplates produced in the RT reaction are then amplified in a PCRamplification method.

Certain embodiments of the present invention also involve sequencingparticular regions of HIV pot. The present inventors targeted variousregions of HIV pol, including particular 1.57 kb and 2.1 kb regions. Toattempt to locate mutations located at the 3′ end of HIV pol, primerswere designed and tested to amplify a 2.1 kb region containing theentire coding region of protease and reverse transcriptase enzymes fromHIV pol. This assay will be used to amplify and sequence the AIDSClinical Testing Group (ACTG) 320 study, which will document themutation patterns and clinical relevance of mutations in HIV pol inresponse to multi-drug therapy (protease and reverse transcriptaseinhibitors). In addition to the 1.57 kb region, an additional 3′ RTregion (approximately 0.7 kb) was targeted for amplification andsequencing. These two amplification and sequencing protocols cover theentire 2.1 kb of HIV protease and reverse transcriptase for the ACTG 320study.

Novel PCR primers were developed and used to amplify the 0.7 kb, 1.57kb, or 2.1 kb regions of HIV pol. The unique PCR primers hybridize tohighly conserved regions of pot. According to certain embodiments, thePCR application employs a hot start enzyme, such as AmpliTaq Gold. Thenovel HIV pol amplification primers are: 0.7 kb primers-GGACTGTCAATGACATACAGAAGTTAGTGG, (SEQ ID NO:3) andGGTTAAAATCACTAGCCATTGCTCTCC; (SEQ ID NO:4) 1.57 kb primers-GGAAAAGGGCTGTTGGAAATGTG, (SEQ ID NO:1) andGGCTCTTGATAAATTTGATATGTCCATTG, (SEQ ID NO:2) 2.1 kb primers-CTCATGTTCATCTTGGGCCTTATCTATTC, (SEQ ID NO:13) and eitherGCCAGGGAATTTTCTTCAGAGCAG, (SEQ ID NO:12) or GGCCAGGGAATTTTCTTCAGAGC.(SEQ ID NO:14)

According to certain embodiments, sequencing procedures can employ oneor more novel sequencing primers specific for the amplified HIV polfragments. The novel HIV pol sequencing primers are: AGCCAACAGCCCCACCAG,(SEQ ID NO:5) CCATCCCTGTGGAAGCACATTG, (SEQ ID NO:6)GTTAAACAATGGCCATTGACAGAAGA, (SEQ ID NO:7) GGAACTGTATCCTTTAGCTTCCC, (SEQID NO:8) AATGCATATTGTGAGTCTG, (SEQ ID NO:9) GAAGAAGCAGAGCTAGAACTGGCAG,(SEQ ID NO:10) and AAGAAGCAGAGCTAGAACTGGCAGA. (SEQ ID NO:11)

The following sequencing primers are also included in sequencingreactions, where appropriate: GGGCCATCCATTCCTGGC, (SEQ ID NO:15)TGGAAAGGATCACCAGCAATATTCCA, (SEQ ID NO:16) andCTGTATTTCTGCTATTAAGTCTTTTGATG. (SEQ ID NO:17)

To sequence the 0.7 kb HIV pol region one can use the primers:AATGCATATTGTGAGTCTG, (SEQ ID NO:9) and either GAAGAAGCAGAGCTAGAACTGGCAG,(SEQ ID NO:10) or AAGAAGCAGAGCTAGAACTGGCAGA. (SEQ ID NO:11)

Because these latter two sequencing primers appear to work equally welland provide similar results they can be used interchangeably. To obtainthe sequence of the 0.7 kb region, one of the two sequencing primers isadded to an aliquot of the 0.7 kb amplified target sequences and thesecond sequencing primer is added to a separate aliquot and thesequencing reaction is completed as described. The sequencing resultsfrom these two separate sequencing experiments are then combined andanalyzed to provide the sequence for the 0.7 kb region.

Seven sequencing primers can be used to sequence the 1.57 kb HIV polregion: AGCCAACAGCCCCACCAG, (SEQ ID NO:5) CCATCCCTGTGGAAGCACATTG, (SEQID NO:6) GTTAAACAATGGCCATTGACAGAAGA, (SEQ ID NO:7)GGAACTGTATCCTTTAGCTTCCC, (SEQ ID NO:8) GGGCCATCCATTCCTGGC, (SEQ IDNO:15) TGGAAAGGATCACCAGCAATATTCCA, (SEQ ID NO:16) andCTGTATTTCTGCTATTAAGTCTTTTGATG. (SEQ ID NO:17)

The procedure is the same as for the 0.7 kb region except that sevenseparate sequencing reactions, one for each sequencing primer, areperformed and the seven sets of sequencing data are combined foranalysis.

To sequence the 2.1 kb region, both the 0.7 kb sequencing primers (SEQID NO:15, and 16 or 17) and the 1.57 kb sequencing primers (SEQ ID NO:5,6, 7, 8, 9, 10 and 11) can be used in nine separate sequencingreactions. The resulting nine sets of sequencing data are combined foranalysis.

According to certain embodiments of the sequencing procedure, the newdye-terminator chemistries (dRhodamine and Big-Dye) were employed inplace of dye-labeled primers. The new dye chemistries allow for moreeven incorporation of nucleotides and a much improved signal to noiseratio over the rhodamine terminators. The new Big-Dye terminator (U.S.Pat. No. 5,800,996) was chosen over the dRhodamine terminators becauseof the increased signal strength and better signal to noise ratio. Thisallows for a faster throughput for sequencing with increased dataquality.

In certain embodiments, the target nucleic acid sequences areautomatically analyzed by software that was developed for assigning anHIV genotype. The software incorporates two new features in thebasecalling function: using known features of the sequences aspreviously determined from a set of standards (Conrad et al., 1995) andusing the base identified on the complimentary strand to confirm thebasecall. The use of experienced basecalling algorithms dramaticallyreduces the need for manual editing. The assembly of the basecallingprimary data into a contiguous sequence is performed in a batch-wisemanner by the software. The software then compares the derived sequenceto a known HIV reference and table of known resistance mutations forgenotypic assignment. Positions are reported that either differ inassignment by each of the sequence segments, differ from the HIV“wild-type” reference, or are found in the table of sequence mutationsknown to be correlated with drug resistance. This system can be coupledwith the use of a sequence database for higher level analysis and datamanagement.

The following examples are intended to be purely exemplary of theinvention and are not intended to be limiting.

WORKING EXAMPLES

The following protocols were used with the 0.7 kb, 1.57 kb and 2.1 kbregions of HIV pot. All method steps and reagents remained constant,including the primer concentration, except that the specific amplifyingand sequencing primers varied based on the HIV pol region beinganalyzed.

Example 1 RNA Preparation

Plasma samples, either freshly obtained or thawed at room temperaturewere vortexed for 3-5 seconds at medium to low speed and then brieflycentrifuged to collect the specimen in the bottom of the tube. One halfml aliquots of plasma were placed into 1.5 ml microfuge tubes with snaptops, transferred to a centrifuge pre-chilled to 40° C. (Heraeus 17RS,Biofuge 22 R, Biofuge 28RS or Beckman Centrifuge GS-1 5R or equivalent),and centrifuged for 1 hour at 21,000 to 25,000×g at 4° C. in apre-chilled rotor. After centrifugation, the supernatant was carefullyremoved and discarded. The pellet was resuspended in 600 μl lysis bufferand vortexed for 3-5 seconds at medium to low speed followed byincubation at room temperature for 10 minutes.

To precipitate the viral RNA, 600 μl of room temperature 100%Isopropanol was added and the capped tube was vortexed for 5-10 secondsat medium to low speed. The tube was then centrifuged in amicrocentrifuge at maximum speed (at least 12,500×g) for 15 minutes atroom temperature. The resulting supernatant was removed and discarded.The tube was then recentrifuged for 5-10 seconds and the residual liquidwas removed with a fine pipette tip. It is important to remove as muchliquid as possible.

The pellet was washed using 1.0 ml 70% ethanol, prechilled to 4° C. Thetube was vortexed for 3-5 seconds to resuspend the pellet, thencentrifuged in a microcentrifuge at maximum speed (at least 12,500×g)for 5 minutes at room temperature. Again the resulting supernatant wasremoved and discarded and the tube centrifuged for an additional 5-10seconds to allow the removal of the residual liquid. To minimize RNAdegradation samples should be kept on ice unless otherwise noted.

The washed RNA was resuspended in specimen diluent (10 ng/ml polyrA inRNase-free water) using either 50 μl if the viral load is either knownor expected to be less than 10,000 copies/ml or 100 μl if the viral loadis greater than 10,000 copies/ml. The sample was vortexed at medium tolow speed for 10 seconds then centrifuged for 5-10 seconds in amicrocentrifuge to collect the liquid to the bottom of the tube. Thepurified RNA was then used immediately as the initial template in thefollowing two-step RT-PCR or stored at −70° C.

Example 2 Two-step, One Tube RT-PCR

The RT and PCR procedures of the instant invention are performed in thesame tube but in two different steps. First purified RNA, HIV-1 RTBuffer (6.25 μM random hexamers, dATP, dCTP, dGTP and dTTP, all at 2.5mM, and 6.25 mM MgCl₂ in 25 mM Tris-50 mM KCl buffer, ph 8.2), RNaseinhibitor (Perkin Elmer) and Maloney Murine Leukemia Virus (MMuLV)reverse transcriptase (Perkin Elmer) were incubated under conditions toallow cDNA synthesis from the purified RNA template, which results inthe generation of HIV derived double-stranded nucleic acid template.

Then, PCR mix (0.34 μM of each specific PCR primer, dATP, dCTP, dGTP anddTTP, all at 0.093 mM, 253 mM MgCl₂, in 10 mM Tris- 50 mM KCl buffer, pH8.2) and the temperature-stable DNA polymerase AmpliTaq Gold (PerkinElmer) was added to the HIV derived double-stranded nucleic acidtemplate and thermal cycled to produce amplified target sequences.

The two-step RT-PCR procedure is carried out in 0.2 ml tubes. The RTreaction mixture containing 8 μl HIV-1 RT Buffer Mix, 1 μl RNaseinhibitor (20 U/μl), 1 μl MMuLV reverse transcriptase (50 U/μl) and 10μl of purified RNA were placed in tubes, transferred to a thermal cyclerpreheated to 42° C. and cycled as follows: 42° C. for 60 minutes, 99° C.for 5 minutes, and were held at 4° C. to synthesize HIV deriveddouble-stranded nucleic acid templates. These templates were usedimmediately for PCR or stored at −20° C. or below until use.

To each tube was added 29.5 μl HIV PCR mix, including 0.5 μl of each ofthe 5′ and 3′ specific PCR primers (20 pmole/μl), and 0.5 μl AmpliTaqGold (5 U/μl). The final volume for PCR is 50 μl. To amplify the targetsequences, the tubes were placed in a thermal cycler (e.g., Perkin Elmermodel 9600, 9700 or 2400) and cycled as follows:

-   -   One cycle of:        -   95° C. for 10 minutes    -   40 cycles of:        -   95° C. for 15 seconds        -   64° C. for 45 seconds        -   68° C for 3 minutes    -   One cycle of:        -   72° C. for 10 minutes followed by a 4° C. soak.

The specific PCR primers were for the 0.7 kb region:GGACTGTCAATGACATACAGAAGTTAGTGG, (SEQ ID NO:3) andGGTTAAAATCACTAGCCATTGCTCTCC; (SEQ ID NO:4) for the 1.57 region:GGAAAAAGGGCTGTTGGAAATGTG (SEQ ID NO:1) andGGCTCTTGATAAATTTGATATGTCCATTG; (SEQ ID NO:2) and for the 2.1. region:CTCATGTTCATCTTGGGCCTTATCTATTC (SEQ ID NO:13) and eitherGCCAGGGAATTTTCTTCAGAGCAG (SEQ ID NO:12) or GGCCAGGGAATTTTCTTCAGAGC. (SEQID NO:14)

Example 3 Purification and Analysis of the RT-PCR Product

The RT-PCR product was purified by electrophoresis on an agarose gelprior to analysis of the target sequences.

Two hundred microliters of sterile water and all of the RT-PCR product(50 μl) was pipetted onto the top of a Microcon 100 microconcentrator(Amicon) and the microconcentrator was sealed with the attached cap. Themicroconcentrator was centrifuged in a bench top centrifuge at 800-845×g(3000 rpm) for 15 minutes at room temperature. The sample reservoir wasremoved from the vial and it was placed upside down in a new vial, whichwas then centrifuged for 3 minutes at 800-845×g (3000 rpm) to transferthe concentrated amplified target sequences to the vial. To each sampleof amplified target sequence was added 35 μl of sterile water and 5 μlwere analyzed by electrophoresis on an agarose gel. The remainingconcentrated template can be stored at −20° C. for several months.

The amplified target sequences were analyzed on 1% agarose gel (SeakemGTC) containing 0.5 μg/ml ethidium bromide. Five microliters ofconcentrated amplified target sequence in an equal volume of agarose gelloading buffer/dye (40% sucrose w/v, bromphenol blue 0.25% w/v, xylenecyanole FF 0.25% w/., 0.1 M EDTA pH 8.0, 0.5% sodium lauryl sulfate) wasloaded into a gel lane and electrophoresed. In an adjacent lane, 2 μl ofa DNA mass ladder was included as a standard (Low DNA Mass Ladder,GibcoBRL; markers for DNA fragments of 2, 1.2, 0.8, 0.4, 0.2, and 0.1 kbin 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, containing 200, 120, 80, 40, 20and 10 ng DNA/4 μl, respectively). The amplified target sequences wereelectrophoresed at 10 V/cm for 45-60 minutes. Upon visualization underUV light, a band containing the amplified target sequence should be seenat 0.7, 1.57, or 2.1 kb, depending on the PCR primers used, providedthat the starting material contained HIV.

If the intensity of the amplified target sequence band is between theintensities of the 1.2 kb and 0.8 kb standard bands, the amplifiedtarget sequence should be diluted with three parts sterile distilleddeionized water for use in the sequencing reaction. If the intensity ofthe amplified target sequence band is less than that of the 1.2 kb andthe 0.8 kb standard bands, dilute the amplified target sequence with anequal volume of sterile distilled deionized water. If the amplifiedtarget sequence band is less intense than the 0.8 kb standard band,there may not be sufficient amplified target for sequencing.

Example 4 Sequencing of the Amplified Target Sequence (RT-PCR Product)

Depending on the HIV pol region being analyzed, the amplified targetsequences were sequenced in either 2, 7, or 9 separate sequencingreactions, each employing different sequencing primers. For sequencingthe 0.7 kb HIV pol region amplified target sequences, the sequencingprimers were: AATGCATATTGTGAGTCTG, (SEQ ID NO:9) and eitherGAAGAAGCAGAGCTAGAACTGGCAG, (SEQ ID NO:10) or AAGAAGCAGAGCTAGAACTGGCAGA.(SEQ ID NO:11)

For sequencing the 1.57 kb HIV pol region amplified target sequences,the sequencing primers were: AGCCAACAGCCCCACCAG, (SEQ ID NO:5)GGGCCATCCATTCCTGGC, (SEQ ID NO:15) TGGAAAGGATCACCAGCAATATTCCA, (SEQ IDNO:16) CTGTATTTCTGCTATTAAGTCTTTTGATG, (SEQ ID NO:17)CCATCCCTGTGGAAGCACATTG, (SEQ ID NO:6) GTTAAACAATGGCCATTGACAGAAGA, (SEQID NO:7) and GGAACTGTATCCTTTAGCTTCCC. (SEQ ID NO:8)

For sequencing the 2.1 kb HIV pol region amplified target sequences,nine sequencing primers were used, the seven 1.57 kb region sequencingprimers and two of the 0.7 kb region sequencing primers (SEQ ID NO:9 andeither SEQ ID NO:10 or SEQ ID NO:11).

Each of the sequencing primers were diluted in BigDye Terminator ReadyReaction Mix (AmpliTaq Karl 1000-1200 U/ml, rTTH pyrophosphate 125-150U/ml, ddA BigDye Terminator 0.27 μM, ddC Big Dye Terminator 0.41 μM, ddGBig Dye Terminator 0.26 μM, ddT Big Dye Terminator 2.8 μM, dATP 250 μM,dCTP 250 μM, dITP 1250 μM, dUTP 250 μM in 200 mM Tris-HCl, pH 9.05 and 5mM MgCl₂) to a concentration of 0.267 mM. Eight microliter aliquots ofthe diluted amplified target sequences were transferred into each of thetwo, seven or nine sequencing tubes (for the 0.7, 1.57 and 2.1 kbregions, respectively) and 12 μl of one of the two, seven or nineprimer-BigDye Terminator Ready Reaction Mix is added to each ofsequencing tubes. The sequencing tubes are centrifuged briefly in amicrocentrifuge to collect the reagents at the bottom of the tube.

The sequencing reaction tubes are then thermal cycled in either a PE9600, 9700, or 2400 thermal cycler as follows: 25 cycles of 96° C. for10 seconds, 50° C. for 5 seconds and 60° C. for 4 minutes, followed by a4° C. soak. The sequencing tubes should be removed from the thermalcycler within 2 hours and either processed or wrapped in foil and frozenimmediately at −20° C.

For each reaction, a 1.5 ml microcentrifuge tube was prepared containing2 μl 3M sodium acetate (NaOAc), pH 4 and 50 μl 95% ethanol and theentire 20 μl sequencing reaction was transferred to the tube. Each tubewas then vortexed 3-5 seconds, placed on ice for 10 minutes andcentrifuged at maximum speed 12500×g (15000 rpm) for 30 minutes in atable top centrifuge, to pellet the sequencing products. The supernatantwas removed and discarded and the pellet was washed with 250 μl of cold70% EtOH, vortexed for 5 seconds and centrifuged for 5 minutes in atable top microcentrifuge at maximum speed 12500×g (15000 rpm). Thesupernatant was removed and the pellet was briefly dried in a pre-heated95° C. heat block for 1-2 minutes. These pellets can be stored at −20°C., for at least 6 months.

The sequence products were analyzed by resuspending each pellet with 5μl of the loading buffer-formamide (1 part recrystallized formamide in 5parts loading buffer −25 mg/l blue dextran, 10 mM EDTA), vortexing 3-5seconds at medium speed, and centrifuging 3-5 seconds at high speed thesamples to bring all the liquid to the bottom of the tube. The sampleswere heated at 95° C. for 2 minutes to denature the sequencing productsand electrophoresed on a sequencing gel at 1500 V.

The sequencing gels were analyzed to determine the target sequences,which were then compared with the “wild-type” HIV pol gene sequence. Allmutations detected were compared with known drug resistance mutations.

The amplification and sequencing methods were used with primers specificfor the 1.57 kb region of HIV pol using plasma samples form patientsknown to be infected with HIV. In one example, PE505, a total of 118nucleotide variations were identified in the 1.57 kb pol region, ofwhich 6 correlated to known drug resistance mutations, 5 in the viralprotease and one in RT.

The sequence of the 1.57 HIV pol region, for clinical sample PE 505 isshown below. At several nucleotides two bases were detected, indicatingthat this sample probably contained more than one HIV quasi-species.CCTCARATCACTCTTTGGCAACGACCAMTAGTCACAATAAAGATAGGGGGGCAATTAAAGGAAGCTTTATTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATGAATTTGCCAGGAAAATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGRTACTCATAGAAATCTGTGGACATAAAGCTATAGGTACAGTATTARTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAACTTGGGTGCACTTTAAATTTTCCTATTAGTCCTATTGAAACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAAAAATTGGACCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAGATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGAYTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAAAAGAAGAAATCAGTGACAGTACTGGATGTGGGTGATGCATATTTCTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCGGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGAGCAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATGGTTATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGAMAACATCTGTTGAAGTGGGGATTTACCACACCAGACAAAAAACATCAGAAGGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGAGCTGCCAGAAAAAGACAGCTGGACTGTCAATGACATACAGAAGTTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATCCAGGAATTAAGGTAAAGCAATTATGTAGACTCCTTAGGGGAGCCAAAGCACTCACAGAAGTAATACCACTAACAAAGGAAGCAGAGATRGAACTGGCAGAAAACAGGGAGATTCTAAAAGAGCCAGTACATGGAGTGTA TTATGA

-   -   R—G or A residues were detected at this position    -   Y—C or T residues were detected at this position    -   M—A or C residues were detected at this position

REFERENCES

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1-42. (Canceled)
 43. A method of identifying mutations associated withdrug resistance in an HIV quasi-species comprising: a) preparing nucleicacid from a human body fluid sample; b) preparing double strandednucleic acid template from HIV RNA in the nucleic acid; c) amplifying aregion of HIV pol from the double stranded nucleic acid template toobtain an amplification product; d) sequencing the amplification productusing one or more specific sequencing primers; and e) identifyingmutations associated with drug resistance in an HIV quasi-species.